Project Summary The regulation of the transcription factor Nuclear Factor Kappa B (NF-kB) by its inhibitor, IkBa, is critical for normal cell growth and misregulation is associated with many diseases. The structure of the complex has been elucidated by x-ray crystallography (Huxford et al. 1998;Jacobs and Harrison 1998) and the interface between the two proteins is comprised of interactions between all six beta hairpin loops of IkBalpha. Attempts to crystallize free IkBa were unsuccessful and amide hydrogen exchange experiments revealed that nearly all of the first, fifth and sixth ankyrin repeats exchanged within a few minutes suggesting they were somehow unstructured (Cray Hughes et al. 2004). The fifth and sixth beta-hairpins show a marked decrease in solvent accessibility upon binding to NF-kB that can not be explained simply from protection at the interface. At first, we interpreted these results as indicating that IkBa folds upon binding, but circular dichroism experiments showed no increase in secondary structure when IkBalpha bound to NF-kB (Croy Hughes et al. 2004). Taken together the results suggest that free IkBalpha is somehow dynamic and perhaps molten globular and that it folds upon binding but the folding is more a decrease in the ensemble of states rather than formation of new secondary structure. The best way to probe such an event is by backbone dynamics experiments. The Specific Aims are: AIM 1: Resonance Assignments of Free IkBa. Uniformly 13C, 15N double-labeled protein and deuterated samples as well as selective labeling will be used. Progress on the assignments has been made based primarily on the TROSY-HNCA and HSQC-NOESY-HSQC spectra of 0.2 mM wild type IkBa, and we now have several stabilized mutants that will be assigned simultaneously. AIM 2: Elucidation of the Dynamics of IkBa in Solution. Standard R1, R2 and heteronuclear NOE experiments as well as rcCPMG experiments will be used. AIM 3: Elucidation of the backbone dynamics of Stabilized Mutants of IkBa. Mutations that restore the consensus sequence for stable ankyrin repeat proteins increase stability by 5 kcal/mol over wild type. The results from these experiments will show how dynamics relates to stability in ankyrin repeat proteins. AIM 4. Compare the backbone dynamics of free and NF-kB-bound IkBa. Relevance to human health This project will help understand the regulation of the NF-kB signaling pathway that is critical for human health and is misregulated in diseases such as cancer, arthritis, asthma, diabetes, AIDs and viral infections.